Mutation Scanning and Genotyping by High-Resolution DNA Melting Analysis

1
Mutation Scanning and Genotyping by
High-Resolution DNA Melting Analysis

• Carl Wittwer, MD, PhD
• Professor of Pathology
• University of Utah

2
High-Resolution DNA Melting

• Scanning PCR products
• Mutation Screening
• HLA Matching
• Genotyping
• Hybridization Probes
• Unlabeled Probes
• Small Amplicons
• Simultaneous scanning and genotyping

3
Amplification Analysis
Temperature
0
5
10
15
20
Time (min)
4
Melting Curves for Different Products (SYBR Green
I)
5
Heterozygote Amplification
Two Homoduplexes
Two Heteroduplexes
6
Heterozygote Amplification
Two Homoduplexes
Two Heteroduplexes
7
100 bp Product
C/C Homozygote C/G Heterozygote C/T
Heterozygote C/A Heterozygote

• Homozygotes are easily distinguished from
  heterozygotes
• Different heterozygotes trace unique melting
  paths

8
SNP Typing in Long Amplicons
CC
SNP typing on a 544bp amplicon. (SNP is in the
low temperature domain)
TC
100
80
60
TT
Fluorescence (Normalized)
40
20
0
85
82
84
83
Temperature (C)
Clin Chem. 2003 Jun49(6 Pt 1)853-60.
9
High-Resolution DNA Melting

• Scanning PCR products
• Finding heterozygotes among homozygotes
• Genotyping alleles
• Complete genotyping of wild type, heterozygous,
  and homozygous variants.

10
Mutation Scanning
2 (716)
3 (133)
4 (133)
5 (207)
6 (263)
1
Typical Mutation Distribution
11
Scanning by Heteroduplex Separation
Temperature Gradient Capillary
Electrophoresis (TGCE)
Denaturing High Pressure Liquid
Chromatography (DHPLC)
12
Mutation Scanning Technologies

• All use PCR first

High-Resolution Melting
Electrophoresis
Load on matrix
dHPLC
Mass Spec
Arrays
Clean up Enzymatic reactions Clean up
Sequencing
13
Scanning by High-Resolution Melting

• Closed-tube
• dsDNA dye before PCR
• No processing, additions, or separations
• No exposure to the environment
• Rapid
• 1-2 min for single samples
• 5-10 min for 96/384 samples
• Non-destructive
• Downstream processing if necessary

14
High-Resolution Melting Platforms(Idaho
Technology)
LightScanner
15
High-Resolution Melting Analysis
16
Melting Analysis
17
Melting Analysis
18
Melting Analysis
19
Use of a DNA toolbox as a model system for
mutation scanning

• Highsmith et al., Electrophoresis (1999), 20
  188-1194
• Constructed plasmids of 40, 50, and 60 GC
  content
• with A, C, G, or T at one position
• PCR primers on each side spaced 50 bp apart

• ? ? ? ? ?

X
? ? ? ? ? ?
20
HR-1

• Sensitivity and Specificity
• Dependence on Product Size
• N1248, each instrument
• Three different targets
• All possible SNPs and WTs

LightScanner
Clin Chem. 2004501748-54.
21
Sensitivity and Specificity(40 GC template)
22
Sensitivity and Specificity(50 GC template)
23
Sensitivity and Specificity(60 GC template)
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Missed Heterozygotes
Homozygous Base A C G
T Heterozygous Base cgt agt act
acg cgt agt act acg cgt agt
act acg cgt agt act acg cgt
agt act acg
bp 400 500 1000 400 800
a false negativea correct calls
40 GC
50 GC
25
Ability to detect allele fractions other than
50 (300 bp product)
100 95 90 75 50
26
Scanning by MeltingApplications

• c-kit (GIST tumors) - Am J Clin Path, 122206-16
  (2004)
• MCAD Mol Genet Metab, 82112-20 (2004)
• SLC22A5 (Urea cycle) Hum Mutat, 25306-13
  (2005)
• BRAF (melanoma) Hum Pathol, 36486-93 (2005)
• Cystic fibrosis Am J Clin Path,124330-8
  (2005)
• Studies in progress
• PNH (Paroxysmal nocturnal hemoglobinurea)
• HHT (Hereditary hemorrhagic telangiectasia)
• Galactosemia

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GIST Mutation Detection by Melting Analysis (exon
11)
Normal
c-kit mutation
28
HHT Exon ScanningEng (exon 2 274 bp)
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Exon Scanning (Difference Plot)Eng (exon 2)
274 bp
Fluorescence Difference
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Differences between Heterozygotes
Clin Chem 511295-8 (2005)
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Transplantation Matching
HLA A1, A27, B17, B44, C2, C5, DR1, DR4 1,200
per individual
32
HLA-B
Exon 3
Exon 2
33
HLA-C
Exon 2
Exon 3
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HLA Matching, not Typing

• Tissue Antigens. 2004 Aug64(2)156-64 .
• Applications
• Living related donors
• Unrelated bone marrow transplantation
• ?? Phenotype/Genotype Correlation ??
• ?? Identity ??

35
Genotyping Methods

• Open-tube (processing after amplification)
• Conventional methods (SNE)
• Arrays
• Mass spectrometry
• Closed-tube (real-time)
• Allele-specific
• Melting methods

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Closed-Tube GenotypingPopular Probe Designs
One probe needed for each allele
37
Genotyping by Melting
Adjacent Hybridization Probes (HybProbes) Am J
Pathol. 19981531055-61
One probe pair distinguishes many alleles
38
Dynamic Dot Blot for Allele Analysis
(Heterozygote)
Mutation Probe
Anchor Probe
Match
Mismatch
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Factor V Leiden
Clin Chem 1997 43 2262 - 2267
40
Genotyping by Melting
Adjacent Hybridization Probes (HybProbes) Am J
Pathol. 19981531055-61
Single Probes (SimpleProbe) Anal Biochem.
200129089-97
41
Different Single Labeled Probes(CFTR)
?F508
WT
F508C
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Genotyping by Melting
HybProbes Am J Pathol. 19981531055-61
Single Probes (SimpleProbe) Anal Biochem.
200129089-97
Unlabeled Probes (dsDNA dye) Clin Chem.
2004501328-35
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Unlabeled Probe Genotyping(Factor V Leiden)
Clin Chem 2005 51 1770 - 1777
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Wild Type Heterozygous Mutant Homozygous Mutant
Automatic Clustering
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Genotyping by Melting
HybProbes Am J Pathol. 19981531055-61
SimpleProbe Anal Biochem. 200129089-97
Unlabeled Probes (dsDNA dye) Clin Chem.
2004501328-35
Amplicon Melting Anal. Biochem. 1997245, 154-60
(SYBR Green I) Clin Chem. 200349732-9 (LCGreen)
One amplicon distinguishes many alleles
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Amplicon Melting(Factor V Leiden)
Temperature (C)
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Distinguishing all 4 homoduplexes and all 6
heteroduplexes
A/A C/C G/G T/T
A/C A/G A/T C/G C/T G/T
48
Most, but not all SNP homozygotes can be
distinguished (Clin Chem. 2004501156-64)
Human SNP Percentage
(C)
49
H63D Genotype Melting Curves
No Wild Type Addition
With Wild Type Addition
(Optimal Wild Type fraction ?)
Anal Biochem 2005, in press
50
Genotyping by Melting with dsDNA dyes

• No covalent labels
• Multiplexing by temperature, not color

51
Comparison of Methods for Real-Time SNP Typing
Probes
Modifications
Method
0 1 1 2 2 2 2 2
Amplicon Melting Unlabeled Probe Single
Probe HybProbe TaqMan Beacons Scorpions MGB TaqMan
0 0 1 1 2 2 3 3
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HFE Mutations
Small Amplicon Melting
Unlabeled Probe
T189C
C282Y (G845A)
H63D (C187G)
S65C (A193T)
53
HFE Genotyping
54
HFE Genotyping
55
HFE Genotyping
56
HFE Genotyping
57
Simultaneous Unlabeled Probe and Amplicon
Melting(Factor V Leiden)
Wild Type Heterozygous Mutant Homozygous Mutant
Temperature (C)
58
Scan Genotype at once
59
CFTR exon 10 scanning and genotyping
-dF/dT
Temperature
60
Gene Analysis
Reported mutations
2 (716)
3 (133)
4 (133)
5 (207)
6 (263)
1
61
Eliminate 99 of sequencing?
Scanning of PCR fragments for variants
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High-Resolution Melting Analysis

• Dyes
• SYBR Green I (1997)
• LCGreen high-resolution dyes
• LCGreen I (2003)
• LCGreen PLUS (2005)
• Instruments
• Single sample HR-1 (2003)
• 96/384 LightScanner (2005)

63
Thanks

• University of Utah
• Mathematics
• Bob Palais
• Pathology
• Luming Zhou
• Gundi Reed
• Rob Pryor
• Josh Vanderstein
• Joe Holden
• Phil Bernard
• ARUP
• Mark Herrmann
• Michael Liew
• Mike Seipp
• Becky Margraf
• Bob Chou
• Idaho Technology
• Virginie Dujols

NIH Whitaker Foundation State of
Utah University of Utah ARUP Idaho
Technology Roche